The present invention relates to the bioreversible functionalization of phosphate or phosphonate groups of biologically active compounds.
The present invention relates more particularly to phosphotriester-type biologically active compounds bearing phosphate or phosphonate groups which are protected by protecting groups that are bioreversible in an intracellular medium.
Compounds bearing a phosphate or phosphonate group have a negatively charged ionic nature and a physiological pH. As a result, the therapeutic activity of such compounds is limited by the low diffusion of negatively charged compounds across biological lipid membranes. Moreover, compounds bearing phosphate groups are readily dephosphorylated by the action of phosphatase enzymes in the blood or on cell membranes, which enzymes dephosphorylate substrate compounds. In general, charged phosphate or phosphonate compounds are poorly absorbed via oral administration, and do not diffuse efficiently across cell membranes or even the cerebral barrier, which are lipidic in nature.
Certain compounds, such as nucleoside derivatives or analogs, are active agents that are administered in non-phosphorylated form, but are phosphorylated in vivo in the form of metabolic monophosphate or triphosphate to become active.
Thus, nucleoside derivatives having antitumor activity, such as 5-fluorouridine, 5-fluoro-2xe2x80x2-deoxyuridine or 1-O-D-arabinofuranosylcytosine, exert their activity in phosphorylated form.
Similarly, in order to exert their antiproliferative activity, certain nucleoside or phosphononucleoside analogs need to be phosphorylated into the corresponding triphosphate thereof by cellular or viral enzymes; this triphosphate is then capable of inhibiting the viral and/or cellular polymerases.
Among the various structural classes of antiviral agents, 2xe2x80x2, 3xe2x80x2-dideoxynucleosides are among the most effective compounds in the treatment of AIDS. However, these nucleoside analogs must undergo a biotransformation by cell kinases in order to exert their activity on the replication of HIV, the etiological agent of AIDS. This metabolization occurs via the dideoxynucleoside 5xe2x80x2-monophosphate and then the 5xe2x80x2-diphosphate to lead to the 5xe2x80x2-triphosphate, which is an inhibitor of HIV reverse transcriptase and which thereby interferes with the biosynthesis of viral DNA.
Despite their great therapeutic potential, 2xe2x80x2,3xe2x80x2-dideoxynucleosides suffer from limitations, in particular the low metabolizability of some of them by kinases into triphosphate. 2xe2x80x2,3xe2x80x2-Dideoxyuridine 5xe2x80x2-triphosphate, for example, is an excellent inhibitor of reverse transcriptase (Z. Hao et al., Proc. Am., Assoc. Cancer Res., 1988, 29, 348, E. Matthes et al., Biochem. Biophys. Res. Commun, 1987, 148, 78-85). However, the nucleoside thereof is able to inhibit the replication of HIV in vitro. Studies have shown that this result is linked to the low metabolizability of the nucleoside into its monophosphate by cell kinases (Z. Hao et al. Mol. Pharmacol. 1990, 37, 157-153).
Thus, AZT is successively metabolized into the triphosphate thereof (AZTP), which is a potent inhibitor of HIV reverse transcriptase. Similarly, Acyclovir (ACV) is converted into the triphosphate thereof (ACVTP) which selectively inhibits herpesvirus DNA polymerase. The first step in the activation of the nucleosides (Nu) consists of a monophosphorylation, leading to the corresponding monophosphate (NUMP). It is this first step which is the most selective.
In order to circumvent this key step of enzymatic monophosphorylation, it has already been proposed to administer NuMPs directly, but their use for therapeutic purposes was contraried by the abovementioned limitations and drawbacks.
Compounds bearing a phosphate or phosphonate group have a negatively charged ionic nature at physiological pH. The therapeutic activity of such compounds is consequently limited, on account of the low diffusion of negatively charged compounds across biological lipid membranes. In particular, charged compounds do not diffuse efficiently across cell membranes, or indeed across the cerebral barrier, which are lipidic in nature. Moreover, such compounds are readily dephosphorylated by the action of phosphatase enzymes in the blood or on the cell membranes, which enzymes dephosphorylate the substrate compounds thereof. In general, charged phosphate or phosphonate compounds are poorly absorbed via oral administration.
It has been sought to convert mononucleotides into neutral phosphotriesters capable of crossing the cell membrane and of intracellular delivery of the corresponding mononucleotide phosphotriester (NUMP). Such an approach has been adopted by various authors for a number of years, but has proved to be disappointing. The derivatives obtained were in general either excessively toxic or of insufficient extracellular stability, and did not in the end result provide any enhancement of the biological activity.
Thus, the use of phosphorylated nucleoside structures comprising bioreversible protecting groups of acyloxymethyl or acyloxybenzyl type has been proposed, for antitumor nucleoside derivatives such as 5-fluorouracil, in WO patents No. 9,008,155 and 9,119,721. However, these compounds are of limited chemical stability, and generate toxic formaldehyde metabolites in vivo. Furthermore., they are sparingly soluble and the yield of their chemical preparation is low.
The aim of the present invention is thus to provide other types of bioreversible groups which may be combined especially with mononucleotide or other structures such that the biological activity thereof is enhanced, in particular as regards compounds derived from or analogous to nucleosides having antiviral activity, and which reversible groups do not have the abovementioned drawbacks.
The present invention proposes to use novel groups, characterized by the presence of xe2x80x94SISxe2x80x94 and/or xe2x80x94S/Cxe2x95x90Z enzymelabile bonds which lead, after enzymatic activation, to the formation of unstable intermediates that selectively release the corresponding monophosphate or monophosphonate.
More precisely, the subject of the present invention is the compound corresponding to the general formula I:
ROxe2x80x94P(xe2x95x90O)(OR)xe2x80x94Nuxe2x80x83xe2x80x83(I)
in which:
R is a radical xe2x80x94(CH2)nxe2x80x94Sxe2x80x94X, where X represents a radical xe2x80x94C(xe2x95x90Z)(Y) or xe2x80x94Sxe2x80x94U, and Z is O or S;
Y and U represent an alkyl, aryl or saccharide radical which is optionally substituted, in particular with an OH, SH or NH group; and
n is equal to 1 to 4, preferably 1 or 2;
Nu is a radical consisting of a residue of a biologically active compound or the dephosphorylated residue of a compound which is biologically active when it bears a phosphate or phosphonate group.
Moreover, the present invention also relates to the compound corresponding to the general formula Ia:
RSxe2x80x94P(xe2x95x90O)(QR)xe2x80x94Nuxe2x80x83xe2x80x83(Ia)
in which:
R is a radical xe2x80x94(CH2)nxe2x80x94Wxe2x80x94X, where X represents a radical xe2x80x94C(xe2x95x90Z)(Y) or xe2x80x94Sxe2x80x94U, and Z is O or S;
Q is O or S;
W is O or S;
Y and U represent an alkyl, aryl or saccharide radical which is optionally substituted, in particular with an OH, SH or NH group;
n is equal to 1 to 4, preferably 1 or 2; and
Nu is a radical consisting of a residue of a biologically active compound or the dephosphorylated residue of a compound which is biologically active when it bears a phosphate or phosphonate group.
When, in the formulas (I) and (Ia), Nu is linked to the phosphorus by a Pxe2x80x94O bond, the compound of formulas (I) and (Ia) according to the invention bears a phosphate group and thus constitutes a phosphotriester compound.
When Nu is linked to the phosphorus by a Pxe2x80x94C bond, the compound of formulas (I) and (Ia) according to the invention bears a phosphonate group.
The mechanisms of bioreversibility of the radicals R take place via enzymatic cleavage of the Sxe2x80x94X or Oxe2x80x94X bonds and release of the (CH2)2xe2x80x94S residues, according to the mechanisms which are illustrated by the examples represented FIG. 1 and FIG. 9.
For Y and U there are especially mentioned, as alkyl group, a C1 to C7 alkyl; as aryl group, phenyl and benzyl radicals, and, as saccharide radicals, glucose, mannose or rhamnose.
In one embodiment, when X represents SU, U preferably represents the radical xe2x80x94(CH2)n1xe2x80x94X1 where X1 represents H, OH, SH or NH2 and n1 is equal to 1 to 4, preferably 1 or 2.
There are especially mentioned the compounds (I) and (Ia) in which R represents xe2x80x94(CH2)2xe2x80x94Sxe2x80x94Sxe2x80x94(CH2)2xe2x80x94OH.
In another embodiment, when X represents xe2x80x94C(xe2x95x90Z)Y, Y appropriately represents CH3 or tBu.
There are especially mentioned the compounds (I) and (Ia) for which R represents xe2x80x94(CH2)nxe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94CH3 or (CH2)nxe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94tBu with n=1 or 2.
In an advantageous embodiment of the present invention, for the compounds (I) and (Ia), there are especially mentioned the compounds for which Nu represents a 5xe2x80x2residue of a natural nucleoside or of a derivative of a natural nucleoside, which is therapeutically active or for which the 5xe2x80x2-(O)-monophosphate or 5xe2x80x2xe2x80x94(C)-monophosphonate is therapeutically active.
These compounds of formulas (I) and (Ia) generally have antiviral or antitumor activity.
The compounds of formulas (I) and (Ia) for which Nu represents a 5xe2x80x2residue of 2xe2x80x2,3xe2x80x2-dideoxynucleoside or 2xe2x80x2,3xe2x80x2-didehydronucleoside are more particularly mentioned.
The compounds (I) and (Ia) for which Nu is a 5xe2x80x2 residue of ddU (dideoxyuridine), ddT (dideoxythymidine), ddC (dideoxycytidine), AZT (3xe2x80x2-azido-2xe2x80x2,3,-dideoxythymidine) and the derivatives thereof, especially those substituted on the pyrimidine base or at 2xe2x80x2 and 3xe2x80x2 of the saccharide ring, are more particularly mentioned among the compounds (I) and (Ia) derived from dideoxynucleosides having antiviral activity.
ddT, ddC or AZT are illustrations of the radicals Nu which represent a 5xe2x80x2 residue of a therapeutically active natural nucleoside derivative.
ddU is an illustration of the radicals Nu which represent a 5xe2x80x2residue of a nucleoside derivative which is only active in phosphorylated form. ddU (dideoxyuridine) is not enzymatically monophosphorylated in vivo. Only the triphosphate thereof is a polymerase inhibitor and imparts antiviral activity thereto.
The compounds for which Nu represents a 5xe2x80x2 residue of the derivatives 5-fluorouridine or 5-fluoro-2xe2x80x2-deoxyuridine or 1-xcex2-D-arabinofuranosylcytosine are especially mentioned among the compounds (I) and (Ia) having antitumor activity. These compounds illustrate the advantage of the functionalization according to the invention in order to circumvent the resistance acquired to certain nucleoside drugs when this resistance is due to a loss of their ability to be monophosphorylated, as is often the case in antitumor chemotherapy.
According to another embodiment variant of the invention, in the compounds (I) and (Ia) the radical Nu represents a nucleoside analog residue such as a carbonucleoside (nucleoside in which the oxygen of the saccharide ring is replaced by a carbon), a phosphononucleoside (nucleoside in which the oxygen at 5xe2x80x2is replaced by a carbon) or a purine- or pyrimidine-based derivative of acyclonucleoside type, that is to say one which contains no saccharide ring, such as ACV (aciclovir), or a methoxyalkylpurine or pyrimidine radical of formula CH2xe2x80x94O-alkylpurine or -pyrimidine.
The compounds (I) and (Ia) for which Nu represents a methoxyalkylpurine or -pyrimidine radical are illustrations of the phosphonate compounds. In the particular case of phosphonylmethoxyalkylpurine or -pyrimidine antiviral compounds, PMEA, HPMPA or HPMPC are especially mentioned, the formulae of which are given in FIGS. 3 and 4.
Thus, the present invention relates in particular to compounds in which Nu is a 3-hydroxy-2-methoxypropylpurine or -pyrimidine radical of formula: xe2x80x94CH2xe2x80x94OCH (CH2OH)xe2x80x94CH2-purine or -pyrimidine or a 2-methoxyethylpurine or -pyrimidine radical of formula xe2x80x94CH2xe2x80x94Oxe2x80x94C2H4-pyrimidine and, for example, the compounds (I) and (Ia) for which Nu is a methoxyethyladenine or 3-hydroxy-2-methoxypropylcytosine radical.
When Nu represents a dephosphonylated residue (dephosphated or dephosphonated) of a molecule which is biologically active when it is in phosphate or phosphonate form, the functionalization according to the invention may enable the physicochemical and biophysical parameters of the said molecule comprising a phosphate or phosphonate group to be modified in general. Compounds (I) and (Ia) may then consist, for example, of a phosphopeptide or phospholipid compound.
When Nu represents a residue of a nucleoside, of a nucleoside derivative or of a nucleoside analog, the latter may be D or L enantiomers.
The compounds according to the invention may be prepared by processes known to those skilled in the art.
In particular, the subject of the present invention is a process for the preparation of the compounds according to the invention, characterized in that a compound of formulas (I) and (Ia) is prepared, in which compound the functional groups of R, and possibly of Nu, are protected by suitable protecting groups, followed by deprotection of the said functional groups of R, and possibly of Nu, in order to obtain the compounds of formula (I) and (Ia).
In particular, a compound of formula (II):
Oxe2x88x92xe2x80x94P(xe2x95x90O)(Oxe2x88x92)xe2x80x94Nuxe2x80x83xe2x80x83(II)
where Nu is possibly protected, is reacted in an appropriate manner with the compound of formula (III):
Xxe2x80x94Sxe2x80x94(CH2)nxe2x80x94OHxe2x80x83xe2x80x83(III)
where X is protected, in order to obtain the said protected compound of formula (I), which is then deprotected.
In a particular embodiment, the reaction between the compounds of formula (II) and (III) takes place in the presence of a condensing agent such as MSNT, in pyridine.
Other preparation processes are illustrated in the examples which follow, in which other characteristics and advantages of the present invention will also appear.